【作者】 郭莉；

【导师】 丘小庆；

【作者基本信息】 四川大学 ， 生理学， 2005， 硕士

【摘要】 金黄色葡萄球菌是引起院内感染和社区感染的重要致病菌之一,耐甲氧西林金黄色葡萄球菌(methicillin-resistent S.aureus,MRSA)的出现和近年来耐万古霉素的金黄色葡萄球菌的出现(Vancomycin-resistent S.aureus,VRSA)正严重威胁着人类健康,因此迫切地需要发展新型的抗菌素。 Ph-SA(Pheromonicin-SA Ph-SA)是本实验室以大肠菌素Ia(colicin Ia)和金黄色葡萄球菌的信息素AgrD1通过分子生物学方法构建的多结构域融合抗菌多肽。AgrD1可以将原本不能识别金黄色葡萄球菌的大肠菌素Ia引导至金黄色葡萄球菌胞膜的亲疏水作用区内,大肠菌素Ia的通道形成结构域就可以循亲疏水作用插入细菌胞膜,形成电压依赖性的离子通道,将细菌杀死。 AgrD是金黄色葡萄球菌密度阈值感应系统(accessory gene regulator system,agr)的信号分子,当其浓度达到一定的阈值,便会与金黄色葡萄球菌胞膜上的受体结合,通过二元信号传导系统启动基因的表达,促进金黄色葡萄球菌大部分毒力因子的分泌,与金黄色葡萄球菌的致病性密切相关。Ph-SA是一个融合蛋白,其结构特点相当于将AgrD1序列的氨基端连接在大肠菌素Ia的羧基端,同天然的AgrD1相比,它对agr系更多还原

【Abstract】 Staphylococcus aureus(SA), a significant human pathogen of nosocomial and community-acquired infection, has developed serious resistance to the most available antibiotics. The emergence of methicillin and vancomycin-resistant SA strains have brought enormous threat to the public health. Thus we have to develop novel antimicrobial agents for prevention and treament of antibiotic-resistant bacterial, especially staphylococcal infections.Pheromonicin-SA (Ph-SA is a peptide consisting of a staphylococcal AgrD 1 pheromone fused to the C-terminus of colicin Ia, a channel-forming bacteriocin had presented remarkable bactericidal activity against methicillin-resistant SA (MRSA). AgrD 1 targeted the channel-forming domain of colicin Ia to the cytoplasmic membrane of SA cells, then the channel-forming domain of colicin Ia inserted into the membrane to form the voltage-activated channel to kill the SA cells.The agr (accessory gene regulator) locus of SA cells is the substantial entity for the adjusting of virulence gene expression. It is a classical two-component quorum sensing regulatory system of prokaryotic cells.Based upon the structure of Ph-SA molecules, then, what kind of functional influence of Ph-SA should act on that agr system if the AgrD 1 epitope of Ph-SA molecules binding to the relative membrane agr receptors?In the present study, SA cells were divided into three groups, controK penicillin-treated and Ph-SA-treated. After SA cells growing in liquid medium about 3 hours, PBS, penicillin and Ph-SA were added into the medium respectively when the turbidity of medium reached 0.2(As95). Growing with agents for one hour, the supernant of medium were collected for the subsequent assays.The amount of delta-toxin from different groups and growth phases was measured by high performance liquid chromatography (HPLC). Influence of Ph-SA on SA exoprotein productions was shown by SDS-PAGE assay and the hemolytic activities of cultured filtrate on human erythrocytes were measured.Significantly, it was observed that the secretion of 5 -toxin in Ph-SA group was less than that of the other two groups at the same growth time(P<0.05), so were the production of exoproteins and proteins in cells in SDS-PAGE assay. These results indicated that Ph-SA molecules inhibited exoprotein production of tested S A cells. In addition the results of hemolytic activities on human erythrocytes were also supported our imaginationToxin production is the main pathogenicity of SA to the host. We suggested that Ph-SA could block the activity of agr system, acting as a pseudo-ligand, which can attenuate the virulence of SA via inhibition of exoprotein production.更多还原